Fig 1: Oxidative stress underlies the ability of therapy-entrained neutrophils to exert cytotoxic phenotypes.(A) Coculture of neutrophils isolated from control- or therapy-treated 4T1 tumor-bearing mice with 4T1 cells (5:1 ratio). Cocultures were treated with phenformin (0.25 mM), verdiperstat (4 nM), or FPS-ZM1 (6 nM) (n = 8 per group). HBSS, Hanks’ balanced salt solution. (B) DHE and (C) DCFDA levels (% positive cells and mean fluorescence intensity) from blood Ly6G+/CD11b+ neutrophils isolated from 4T1 tumor-bearing mice treated with vehicle, polyIC, and/or phenformin (five to eight tumors per group). (D) ROS production from PMA-stimulated neutrophils isolated from 4T1 tumor-bearing mice. Before PMA stimulation, neutrophils were treated with vehicle, MPO inhibitor (MPOi) (verdiperstat) or recombinant SOD3. In vivo ROS levels in (E) 4T1 and (F) AT3 tumor-bearing mice treated with vehicle, phenformin, and/or polyIC (six to eight tumors per group). (G) 4T1 tumor-bearing mice were treated with vehicle, phenformin, and/or polyIC, and cohorts received verdiperstat (5 mg) or DMSO [average tumor volume (mm3) ± SEM (n = 9 to 12 tumors per group)]. (H) Tumor mass and (I) in vivo ROS levels at the endpoint. (J) SOD3 levels produced from tumor lysates or secreted into the conditioned media of empty vector and SOD3-overexpressing 4T1 cells as measured by ELISA (n = 3). (K) 4T1 (EV and SOD3-overexpressing) tumor-bearing mice were treated with vehicle, phenformin, and/or polyIC (average tumor volume (mm3) ± SEM; n = 10 to 12 tumors per group). (L) Tumor mass and (M) in vivo ROS levels at the endpoint. P values were calculated using a one-way ANOVA with a Tukey’s post hoc test [(B), (C), (E), (F), (G) to (I), and (K) to (M)] or a two-tailed Student’s t test [(A) and (J)]. RLU, relative light units.